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Liquid Based Diagnostics
Theresa Boyle, MD, PhD Assistant Member/Molecular
Pathologist Moffitt Cancer Center
Liquid Based Diagnostics
Disclosures/Experience
• No financial disclosures
• Trained to sign-out circulating tumor cell cases
(CellSearch)
• Quality assurance project at initiation of send-out for
blood targeted genetic testing (Biodesix)
• Medical advisor for Moffitt Cancer Center Clinical
Genomics Action Committee with review of
comprehensive blood mutation testing results
(Guardant 360 and FoundationOne)
• Urine translational research (Trovagene)
Liquid Based Diagnostics
• Description and History
• Circulating tumor cells (CTCs)
• Circulating cell-free DNA (cfDNA) from
tumor in plasma and urine
• Serum protein biomarkers
History of Liquid Based Diagnostics
• 1869: CTCs noted in the blood (Thomas Ashworth)
• 1948: Tumor nucleic acid identified in plasma (Mandel P and Metais P.) – Les acides nucleiques du plasma sanguin chez l’homme. CR. Acad Sci. Paris 142:241-243
• 1997: Fetal cfDNA detected in blood (Lo YM) – Later, incidental early detection of cancer in rare cases
• 2004: FDA approved assay to detect CTCs in blood for patients with breast cancer for prognosis; later expanded to prostate and colon cancers (Miller MC, Doyle DG)
• 2014: Comprehensive NGS blood based tumor cfDNA testing first offered commercially (Guardant first, now others)
• 2016: FDA approved cfDNA test for EGFR mutation in blood from patients with lung cancer
Analysis of circulating tumor cells (CTCs)
Lowes LE et al. Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation. Int J Mol Sci. Sep 2016.
Detection of Circulating Tumor Cells
Hayes DF, et al. Circulating Tumor Cells at Each Follow-up Time Point during Therapy of Metastatic Breast Cancer Patients Predict Progression-Free and Overall Survival Clin Cancer Res 2006;12(14): 4218-4224.
de Bono, JS, et al. Clin Cancer Res 2008 Cohen SJ, et al. J Clin Oncol. 2008.
In lung cancer, EMT often decreases EpCAM, which is needed for capture by CellSearch . CTC as a prognostic marker is unclear. Conclusion by Ma X-L et al: “Detection of CTCs in the peripheral blood indicates a poor prognosis in patients with lung cancer.” -Asian Pacific J Cancer Prev, 2012; Metanalysis of 12 articles Conclusion by Chudasama D et al: “The results of this study suggest that the presence of CTCs analyzed by ScreenCell did not necessarily lead to a poorer prognosis in patients with lung cancer after curative surgery.” -Anticancer Res 2017; N=23
Automated Optimization of Ferrofluid Binding Activity
Anti-EpCAM-Ferrofluid binds to CTCs
Exp.1 Analysis of ER-PE conjugated antibody. Approximately 100 fixed MCF-7 cells were spiked into 14ml processing
buffer and processed with the CXC kit with either no antibody or with 450UL NEAT ER-PE conjugated antibody and then
analyzed for ER expression. Twenty five microliters of focusing beads were added to each sample prior to scanning and
CXC control cells were run to confirm processing.
DAPI/CK-FLU CK-FLU DAPI CD45 APC ER-PE
MCF-7 Cells
Minus Ab
MCF-7 Cells
Plus Ab
ER-PE Antibody Expression
Nicolazzo C, Sci Rep 2016
Nicolazzo C, Sci Rep. August 2016
At 6 months, all patients with PD-L1(–) CTCs had obtained a clinical benefit, while all patients with PD-L1(+) CTCs experienced progression of disease.
CB: clinical benefit, PD: progression of disease
PD-L1 status in lung CTCs at baseline, at the first follow-up (3 months of treatment) and at the second follow-up (6 months of treatment).
N=24
Tumor circulating cell free DNA (cfDNA)
• Applications – Identification of
molecular targets – Early Detection – May catch mutations
missed by biopsy due to tumor heterogeneity
– Early Treatment Response
– Monitoring of minimal residual disease
– Evolution of Resistance
Diaz et al. JCO. Feb 2014
Advantages of Blood cfDNA Testing by Lung
Cancer Stage and Line of Therapy
• Early-Stage
– Can screen for adjuvant therapy trial (ex. Alchemist) • Allows for tissue block preservation for future testing including clinical
trials
– If EGFR mutation detected and recurrence, patient more likely to return for targeted therapy
• Advanced Stage
– 1st Line: Targeted methods allows for rapid results for clinical trial enrollment /targeted therapy • FDA approved therapies for EGFR (TKI), ALK or ROS1 Fusions (Crizotinib)
• BRAF mutation = clinical trial or BRAF and MEK inhibitor (ex. dabrafenib and tremetinib)
• KRAS Mutation = Patient may be eligible for clinical trial and not benefit from additional mutation testing
– Later Line: • Detect resistance mutations:
– EGFR T790M = TKI Inhibitor Resistance – Administer 3nd Generation TKI Inhibitor Therapy
– Other resistance mutations, such as EGFR C797S, PIK3CA, MET, ALK,..
• RET (RET inhibitor), ERBB2 (afatinib or trastuzumab)
• NTRK fusion = clinical trial
• Other mutations = clinical trial
Advantages of Blood cfDNA Testing by Lung
Cancer Stage and Line of Therapy
1st line Later line Early
stage
Digital Droplet PCR Technology
Droplet digital PCR improves precision, sensitivity, and reproducibility as compared to previous generation technology
References: Chen et al, Molecular Therapy Nucleic Acids (2013) 2, e109; doi:10.1038/mtna.2013.28
Better survival with lower ctDNA, quantity associated with stage
Schwaederle M et al. Use of liquid biopsies in clinical oncology: pilot experience in 168 patients. May 2016
PFS of p-EGFR and t-EGFR mut+ patients
TS Mok et al: Fast Act 2 – study ASCO 2013 Annual Meeting
Urine testing for ctDNA
• ctDNA released in blood passes to urine – 10ml blood=60 ng DNA
– 10ml urine=143 ng DNA
– Quality?
• Urine easy to collect in large volumes at home
Trovagene website
Siravegna and Bardelli, 2017
Liquid Based Diagnostics: Proteomics • Classical serum protein biomarkers: PSA, CEA, CA-125
• Mass spectrometry can analyze multiple proteins at one time – In lung cancer, VeriStrat good results associated with better prognosis than Veristrat poor
(elevated acute-phase reactants, such as serum amyloid A and CRP)
• Taguchi F et al. J Natl Cancer Inst. 2007; Milan E et al. J Proteomics. 2012
Prognostic, If “Veristrat Poor,” may be more important to consider alternative options to standard chemotherapy, such as clinical trials and immunotherapy
Grossi F et al. Serum proteomic test in advanced non-squamous NSCLC. Br J Cancer. 2017.
Roadmap for Lung Cancer Genetic
Testing: Turn Around Time Matters* Turn 1: “Ultra-rapid” blood targeted genetic testing
-EGFR/KRAS/BRAF/ALK/(ROS1/RET), 72 hr
-If positive, treat. Confirm in tissue if needed.
-If negative, keep hunting.
Turn 2: “Rapid” Tissue Targeted Biomarker Testing, ≤5 days from receipt
in lab after tissue collection/dx (days-weeks)
-If positive, treat.
-If negative and advanced stage, keep hunting.
Turn 3: Comprehensive Genomic Profiling of Tissue or Blood: 2 weeks
from receipt in lab after collection/dx (days-weeks)
*“Approximately 1 of 4 patients with lung cancer begin
treatment without mutation information.”
Summary of Liquid Based Diagnostics
Safe, avoid invasive procedures
Fast, 72 hr TAT
Cost-effective
Informative, especially if positive (high specificity)
Preserves tissue for future testing and clinical trials
Easy serial sampling to detect changes over time
Caveats:
Positive: could be from different cancer than expected
Negative: could be false negative due to not enough cfDNA or
mutation not covered by assay
Acknowledgements
– Pathology Team • Dr. Anthony Magliocco (Mentor)
• Dr. Sonar Altiok
• Dr. Dahui Qin
• John Puskas
– Thoracic Oncology Team • Dr. Eric Haura (Mentor)
• Dr. Jhanelle Gray
• Dr. Eric Toloza
– Clinical Genetics Action
Committee • Dr. Howard McLeod
• Kevin Hicks
• Christine Walko
– Industry Partners
• Biodesix
• Guardant
• FoundationOne
• Trovagene
The End. Any Questions?